In Tetrahymena pyriformis, phosphofructokinase (PFK) is membrane-bound. Enzyme activity is solubilized by treatment of membranes with Triton X-100 or by high ionic strength in the presence of a chelator. The solubilized enzyme has an approximate molecular weight of 300,000 daltons. Membrane-bound PFK is inactivated when incubated with MgATP, NaF, and the 100,000 x g supernatant of a crude extract. The supernatant factor needed for inactivation is heat labile, non dialysable, acid precipitable and (NH4)2SO4 precipitable. It has been partially purified by protamine sulfate precipitation, (NH4)2SO4 precipitation, DE-52 column chromatography and gel filtration. The rate of inactivation is dependent on the concentration of the supernatant factor and that of MgATP. Adenosine, phosphate, pyrophosphate, cyclic AMP, or AMP did not replace the requirement for ATP, whereas ADP did. Experiments using radioactive ATP revealed that ATP binds very tightly to a membrane protein that is smaller than undissociated PFK. The inactive enzyme cannot be reactivated by dialysis, gel filtration, or treatment with acid phosphatase, alkaline phosphatase, or snake venom phosphodiesterase. Three protease inhibitors fail to affect the loss of activity. The irreversible ATP-dependent inactivation of PFK may be due to a ligand-induced conformational change of the enzyme, or possibly to a covalent modification of the enzyme.